The three-dimensional structure of the binding pockets of the SP and SK receptors is being defined with respect to their transmembrane alpha-helices and extracellular domains by determining the sites of covalent attachment of a series of SP and SK derivatives containing the photoreactive amino acid p-benzoylphenylalanine spaced at different positions along the amino acid sequences of the two peptides. For each photoaffinity ligand, the amino acid residues of the binding site that becomes covalently labelled are being determined by microsequencing of purified receptor fragments initially located in the primary sequence with the aid of site-specific antibodies andmass spectrometry. A comparative analysis of the similar and distinctive features of these binding domains should provide insight into the molecular basis of peptide selectivity and receptor activation. A related project is determination of the site of attachment of a tritiated photoreactive azide derivative of the nonpeptide SP antagonist CP-96,345. An understanding of the relationship between the sites for peptide agonists and the nonpeptide antagonist may provide insight into the mechanism of the antagonist effects of this compound, and the information to be gained from the project as a whole may advance the rational design of therapeutic agents for clinical entities in which these peptides participate.